Method of treating retained pulmonary secretions

ABSTRACT

A method of facilitating the clearance of retained pulmonary secretions in a subject using lantibiotics is disclosed. The method comprises administering to the lungs of the subject an effective amount of a lantibiotic. The lantibiotic is preferably administered by topically applying it to the respiratory epithelia, such as by generating an aerosol thereof which is then inhaled by the subject. A preferred lantibiotic for carrying out the present invention is duramycin. The method may be used in treating disorders such as cystic fibrosis, chronic bronchitis, and asthma. Also disclosed is a method of combatting tuberculosis comprising administering a lantibiotic to a subject in need of such treatment.

This application is a divisional of prior application Ser. No.08/074,315, filed Jun. 09, 1993, now U.S. Pat. No. 5,512,269, issued onApr. 30, 1996, the disclosure of which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of removing retained mucussecretions from the lungs of a subject by administering a lantibioticsuch as duramycin to the lungs of the subject. The present inventionalso relates to a method of combatting infection by Mycobacteriumtuberculosis.

BACKGROUND OF THE INVENTION

In cystic fibrosis several functions of airway epithelia are abnormal,and deficiencies in both Cl⁻ transport and Na⁺ absorption are welldocumented. See, e.g. Knowles et al., Science 221, 1067 (1983); Knowleset al., J. Clin. Invest. 71, 1410 (1983). It is believed that impairedchloride secretion is one of the causes of the thickened airway surfaceliquid that characterizes cystic fibrosis. This thickened airway fluidcontributes to the recurrent pulmonary infections and loss ofventilatory function that occur in cystic fibrosis. Retained airwaysecretions are also known to contribute to the morbidity of otherpulmonary diseases such as asthma and chronic obstructive pulmonarydisease.

The therapeutic goal in cystic fibrosis and other pulmonary diseases inwhich the water content of the mucus is altered is to remove retainedsecretions from the lungs. For example, the use of aerosolized amilorideto facilitate the removal of retained mucus secretions is described inU.S. Pat. No. 4,501,729. Amiloride appears to block Na⁺ reabsorption byairway epithelial cells, and therefore inhibits water absorption frommucus.

A different therapeutic approach is to increase the water content of theairway surface liquid by modulating the activity of chloride channels.An example of this is the administration of ATP or UTP, which appear toinduce hydrated mucus secretions by stimulating chloride secretion fromrespiratory epithelial cells. See, e.g., C. Stock, Breathing Easier: APromising Treatment for Cystic Fibrosis, Endeavors, 10, 10-11 (Fall1992) (Published by the Office of Research Services, The University ofNorth Carolina at Chapel Hill).

Tuberculosis, while also primarily involving the lungs of afflictedsubjects, is an infectious disease caused by the bacteria Mycobacteriumtuberculosis. While most commonly occurring in the lungs, tuberculosisinfections may occur anywhere in the body and may be widelydisseminated. The development of streptomycin in 1944, isoniazid (INH)in 1952, ethambutol in 1952, and rifampin in 1972 led to a decreasingprevalence of tuberculosis up until approximately 1985, when the numberof reported cases began to increase, Bloom and Murray Science, 257, 1055(1992). Outbreaks of multi-drug resistant (MDR) tuberculosis areoccurring with increasing frequency, and pose a major public healthproblem, Morbidity and Mortality Weekly Report, 41, 5 (1992).Tuberculosis remains the leading cause of death in the world from asingle infectious disease.

SUMMARY OF THE INVENTION

A method of facilitating lung mucus clearance in a subject in need ofsuch treatment is disclosed. The method comprises administering to thelungs of the subject a lantibiotic such as duramycin or apharmaceutically acceptable salt thereof (hereinafter referred to as an"active compound"), in an amount effective to facilitate clearance oflung mucus. The lantibiotic is preferably administered to the lungs bytopically applying the lantibiotic to the respiratory epithelia (e.g.,the nasal epithelia, trachea, and bronchi).

The method of the present invention may further comprise the step ofconcurrently administering a sodium channel blocker such as amiloride tothe subject in an amount effective to inhibit the reabsorption of waterfrom lung mucous secretions.

The method of the present invention may also further comprise the stepof removing retained mucus secretions from the lungs of the subjectprior to the step of administering the lantibiotic so that applicationof the active agent to the respiratory epithelia is facilitated.Alternatively, the present invention may be carried out prophylacticallyon patients such as children afflicted with cystic fibrosis prior tosubstantial airway blockage and decline of respiratory function.

A method of combatting cystic fibrosis in a subject in need of suchtreatment by administering a lantibiotic as given above to said subjectin an amount effective to hydrate lung mucus secretions is oneparticular aspect of the foregoing method.

A method of combatting chronic bronchitis in a subject in need of suchtreatment by administering a lantibiotic as given above to said subjectin an amount effective to hydrate lung mucus secretions is anotherparticular aspect of the foregoing.

A method of combatting asthma in a subject in need of such treatment byadministering a lantibiotic as given above to said subject in an amounteffective to hydrate lung mucus secretions is still another moreparticular aspect of the foregoing.

A second aspect of the present invention is a pharmaceutical compositioncontaining an active compound as disclosed herein, in an amounteffective to facilitate the clearance of lung mucous secretions, in apharmaceutically acceptable carrier (e.g., a sterile liquid or solidcarrier comprised of respirable particles). The pharmaceuticalcomposition may further contain a sodium channel blocker such asamiloride in an amount effective to inhibit the reabsorption of waterfrom lung mucous secretions.

A third aspect of the present invention is the use of an active compoundas disclosed herein for the manufacture of a medicament for theprophylactic or therapeutic clearance of lung mucous secretions in apatient in need of such treatment.

The use of an active compound as disclosed herein for the manufacture ofa medicament for the prophylactic or therapeutic treatment of cysticfibrosis in a subject in need of such treatment is a particular aspectof the foregoing.

The use of an active compound as disclosed herein for the manufacture ofa medicament for the prophylactic or therapeutic treatment of chronicbronchitis in a subject in need of such treatment is another particularaspect of the foregoing.

The use of an active compound as disclosed herein for the manufacture ofa medicament for the prophylactic or therapeutic treatment of asthma ina subject in need of such treatment is still another particular aspectof the foregoing.

A fourth aspect of the present invention is a method of treatingMycobacterium tuberculosis infection, particularly drug resistanttuberculosis infections, in a subject in need of such treatment. Themethod comprises administering to the subject a lantibiotic such asduramycin or a pharmaceutically acceptable salt thereof in an amounteffective to combat M. tuberculosis infections. The method may furthercomprise the step of concurrently administering additionalanti-tubercular agents in amounts effective to combat M. tuberculosisinfections.

A fifth aspect of the present invention is a pharmaceutical compositioncontaining an active compound as disclosed herein, in an amounteffective to combat M. tuberculosis infections, in a pharmaceuticallyacceptable carrier. The pharmaceutical composition may further containadditional anti-tuberculosis agents in amounts effective to combat M.tuberculosis infections.

A sixth aspect of the present invention is the use of an active compoundas disclosed herein for the manufacture of a medicament for theprophylactic or therapeutic treatment of M. tuberculosis infection in apatient in need of such treatment.

Duramycin (also known as PA48009) is one of a group oflanthionine-containing polypeptide antibiotics. Lanthionine-containingpolypeptides are also known as lantibiotics. See generally G. Jung,Angew. Chem. Int. Ed. Engl. 30, 1051-1068 (1991). The ability ofduramycin to increase chloride secretion has been reported. Stone etal., J. Biol. Chem. 259, 2701 (1984); Cloutier et al., Pediatr.Pulmonol. 1(Suppl), 112 (1987); Cloutier et al., Pediatr. Pulmonol.2(Suppl), 99 (1988); Cloutier et al., Pediatr. Pulmonol. 4(Suppl), 116(1989); Cloutier et al., Am. J. Physiol., 259, C450 (1990). The use ofduramycin for facilitating the removal of retained pulmonary mucussecretions has not heretofore been suggested. See M. Roberts et al., J.Pharm. Exp. Ther. 259, 1050, 1058 (1991)("It seems unlikeley, therefore,that the nonspecific effects of duramycin on animal cells can serve inuseful therapeutic strategies.").

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing the amount of liquid recovered from thetrachea in a canine model at baseline (open box) and that recoveredafter nebulization of normal saline vehicle (cross-hatched box).

FIG. 2 is a graph of the weight of filter paper samples taken fromcanine trachea over a 160 minute interval following administration ofeither duramycin or saline control. Animals treated first with vehicleand then with duramycin one week later demonstrated a stable baselineover 160 minutes. Open boxes are saline treatments; filled boxes areduramycin treatments.

FIG. 3 is a graph comparing the amount of surface airway liquidrecovered from the canine trachea model after administration of salinevehicle followed by administration of duramycin one week later, and theamount of surface airway liquid recovered from the canine trachea modelafter administration of duramycin followed by administration of salinevehicle one week later. Filled symbols are duramycin treatments; opensymbols are vehicle treatments; each symbol (circle, square, trianglewith point up, triangle with point right) represents a different animal.

FIG. 4 is a graph comparing the amount of airway surface liquidrecovered from the canine trachea model after duramycin treatment andafter vehicle control treatment. Open box is vehicle; cross-hatched boxis duramycin treatment; n=6 in each group.

FIG. 5 is a graph comparing the growth of Mycobacterium tuberculosis57778 over 12 days in cultures containing duramycin 100 μM (filled box;line 10), duramycin 10 μM (filled box; line 11), duramycin 1.0 μM(filled diamond, line 12), duramycin 0.1 μM (filled diamond, line 13),streptomycin 6 μg/ml (filled triangle; line 14), isoniazid 0.2 μg/ml(open triangle; line 15), rifampin 2 μg/ml (filled circle; line 16),ethambutol 7.5 μg/ml (filled circle; line 17), or no drug ("x"; line18). This strain is streptomycin (S), isoniazid (I), and rifampin (R)resistant, but ethambutol (E) sensitive.

FIG. 6 is a graph comparing the growth of Mycobacterium tuberculosis63169 in cultures over twelve days containing duramycin 100 μM,duramycin 10 μM, duramycin 1.0 μM, duramycin 0.1 μM, streptomycin 6μg/ml, isoniazid 0.2 μg/ml, rifampin 2 μg/ml, ethambutol 7.5 μg/ml, orno drug. Symbols and abbreviations are as given in connection with FIG.5 above.

FIG. 7 is a graph comparing the growth of Mycobacterium tuberculosis65021 in cultures over twelve days containing duramycin 100 μM,duramycin 10 μM, duramycin 1.0 μM, duramycin 0.1 μM, streptomycin 6μg/ml, isoniazid 0.2 μg/ml, rifampin 2 μg/ml, ethambutol 7.5 μg/ml, orno drug. Symbols and abbreviations are as given in connection with FIG.5 above.

FIG. 8 is a graph comparing the growth of drug-sensitive Mycobacteriumtuberculosis in cultures over twelve days containing duramycin 100 μM,duramycin 10 μM, duramycin 1.0 μM, duramycin 0.1 μM, streptomycin 6μg/ml, isoniazid 0.2 μg/ml, rifampin 2 μg/ml, ethambutol 7.5 μg/ml, orno drug. Symbols and abbreviations are as given in connection with FIG.5 above.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention can be used to facilitate (i.e.,enhance, speed, assist) the clearance of mucus secretions from the lungsof a subject in need of such treatment for any reason, including (butnot limited to) retained secretions arising from airway diseases such ascystic fibrosis, chronic bronchitis, asthma, bronchiectasis, andpost-operative atelectasis (plugging of airways with retained secretionsafter surgery).

While applicants do not wish to be bound to any particular theory of theinstant invention, it appears that the clearance of lung mucussecretions is facilitated in the instant invention by the hydrating ofthe lung mucous secretions, with removal of the secretions bymucociliary action then being facilitated.

The present invention is concerned primarily with the treatment of humansubjects but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

Lantibiotics include, but are not limited to, duramycin, nisin, subtilin(Gross et al. Z. Physiol. Chem., 354, 810 (1973)), epidermin (Schnell etal. Nature, 333,276 (1988)), Pep 5 (Sahl, J. Bacteriol., 162, 833(1985)), gallidermin (Kellner et al, Eur. J. Biochem. 177, 53 (1988)),mersacidin, actagardine (Kettenring et al., J. Antibiotics, 53, 1082(1990)), cinnamycin (Kessler et al., Helv. Chim. Acta, 71, 1924 (1988)),duramycin, and ancovenin (Wakamiya et al., Tetrahedron Lett. 26, 665(1985)). These compounds are known or can be made in accordance withknown procedures which will be apparent to those skilled in the art.

The structure of duramycin is known. See Hayashi et al., J. Antibiotics,43, 1421 (1990). Duramycin is available from Sigma Chemical Co. (St.Louis, Mo., USA) as catalog no. D3168, or can be produced in accordancewith known techniques from Streptoverticillium cinnamoneum subsp.azacolutum (NRRL B-1699) (available from the USDA Agricultural ResearchService, Peoria, Ill., USA) in accordance with known techniques. See,e.g., Hayashi et al., supra, Pridham et al., Phytopathology 46, 575-581(1956); Shotwell et al., J. Am. Chem. Soc. 80, 3912 (1958); S. Nakamuraet al. Biochem. 23, 385 (1984).

The active compounds disclosed herein can, as noted above, be preparedin the form of their pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects. Examples of such salts are (a) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b)salts formed from elemental anions such as chlorine, bromine, andiodine, and (c) salts derived from bases, such as ammonium salts, alkalimetal salts such as those of sodium and potassium, alkaline earth metalsalts such as those of calcium and magnesium, and salts with organicbases such as dicyclohexylamine and N-methyl-D-glucamine.

In one embodiment of the present invention, aerosolized duramycin insolution is administered to the lungs of a subject in an amountsufficient to achieve dissolved concentrations of duramycin on theairway surfaces of the subject of from 10⁻⁸ Moles/liter to 10⁻⁵Moles/liter. More preferably, the dosage may be an amount sufficient toachieve dissolved concentrations of duramycin on the airway surfaces ofthe subject of about 10⁻⁷ Moles/liter to 10⁻⁶ Moles/liter.

Sodium channel blockers which may be used in the present invention aretypically pyrazine diuretics such as amiloride, as described in U.S.Pat. No. 4,501,729 (applicant specifically intends the disclosure ofthis and all other patent references cited herein to be incorporatedherein by reference in their entirety). The term "amiloride" as usedherein includes the pharmaceutically acceptable salts thereof, such as(but not limited to) amiloride hydrochloride, as well as the free baseof amiloride. The quantity of amiloride included may be an amountsufficient to achieve dissolved concentrations of amiloride on theairway surfaces of the subject of from about 10⁻⁷ to about 10⁻³Moles/liter, and more preferably from about 10⁻⁶ to about 10⁻⁴Moles/liter.

As noted above, the method of the present invention may also furthercomprise the step of removing retained mucus secretions from the lungsof the subject prior to the step of administering the lantibiotic. Thisfacilitates application of the active agent to the respiratory epitheliaduring the administering step. Such removal of retained mucus secretionscan be carried out by any suitable means, including postural drainage,antibiotic administration (e.g., intraveneous or inhalationadministration of cephalosporin or aminoglycoside antibiotics such asTobramycin), and/or inhalation administration of DNase. In addition, thepresent invention may be carried out on patients such as children priorto decline of respiratory function (e.g., patients essentially free oflung blockage due to retained mucus secretions). Such patients can begenetically predisposed to becoming afflicted with lung disease (e.g.,cystic fibrosis) as hereinbefore described.

The active compounds disclosed herein may be administered to the lungsof a patient by any suitable means, but are preferably administered bygenerating an aerosol comprised of respirable particles, the respirableparticles comprised of the active compound, which particles the subjectinhales. The respirable particles may be liquid or solid. The particlesmay optionally contain other therapeutic ingredients such as a sodiumchannel blocker as noted above, with the sodium channel blocker includedin an amount effective to inhibit the reabsorption of water from airwaymucous secretions.

Particles comprised of active compound for practicing the presentinvention should include particles of respirable size: that is,particles of a size sufficiently small to pass through the mouth andlarynx upon inhalation and into the bronchi and alveoli of the lungs. Ingeneral, particles ranging from about 0.5 to 10 microns in size (moreparticularly, less than about 5 microns in size) are respirable.Particles of non-respirable size which are included in the aerosol tendto deposit in the throat and be swallowed, and the quantity ofnon-respirable particles in the aerosol is preferably minimized. Fornasal administration, a particle size in the range of 10-500 μm ispreferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing anaerosol can be prepared by combining the active compound with a suitablevehicle, such as sterile pyrogen free water. Other therapeuticcompounds, such as a sodium channel blocker, may optionally be included.

Solid particulate compositions containing respirable dry particles ofmicronized active compound may be prepared by grinding dry activecompound with a mortar and pestle, and then passing the micronizedcomposition through a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprised of the activecompound may optionally contain a dispersant which serves to facilitatethe formation of an aerosol. A suitable dispersant is lactose, which maybe blended with the active compound in any suitable ratio (e.g., a 1 to1 ratio by weight). Again, other therapeutic compounds, such asamiloride, may also be included.

The dosage of active compound for prophylaxis or treatment of lungdisease will vary depending on the condition being treated and the stateof the subject, but generally may be an amount sufficient to achievedissolved concentrations of active compound on the airway surfaces ofthe subject of from about 10⁻⁹ to 10⁻³ Moles/liter, and more preferablyfrom 10⁻⁷ to 10⁻⁵ Moles/liter. Depending on the solubility of theparticular formulation of active compound administered, the daily dosemay be divided among one or several unit dose administrations.Preferably, the daily dose is a single unit dose, which is preferablyadministered from 1 to 3 times a week. Treatments may continue week toweek on a chronic basis as necessary (i.e., the active agent can beadministered chronically). Administration of the active compounds may becarried out therapeutically (i.e., as a rescue treatment) orprophylactically, but preferably the compounds are administeredprophylactically, either before substantial lung blockage due toretained mucus secretions has occured, or at a time when such retainedsecretions have been at least in part removed, as discussed above.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a nebulizer. See, e.g.,U.S. Pat. No. 4,501,729. Nebulizers are commercially available deviceswhich transform solutions or suspensions of the active ingredient into atherapeutic aerosol mist either by means of acceleration of a compressedgas, typically air or oxygen, through a narrow venturi orifice or bymeans of ultrasonic agitation. Suitable formulations for use innebulizers consist of the active ingredient in a liquid carrier, theactive ingredient comprising up to 40% w/w of the formulation, butpreferably less than 20% w/w. the carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride. Optional additivesinclude preservatives if the formulation is not prepared sterile, forexample, methyl hydroxybenzoate, antioxidants, flavoring agents,volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol .containing a predetermined metered dose ofa medicament at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which may be delivered by means of aninsufflator or taken into the nasal cavity in the manner of a snuff. Inthe insufflator, the powder (e.g., a metered dose thereof effective tocarry out the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of a powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises from 0.1 to 100 w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofthe active ingredient in a liquified propellant. During use thesedevices discharge the formulation through a valve adapted to deliver ametered volume, typically from 10 to 150 μl, to produce a fine particlespray containing the active ingredient. Suitable propellants includecertain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation mayadditionally contain one or more cosolvents, for example, ethanol,surfactants, such as oleic acid or sorbitan trioleate, antioxidants andsuitable flavoring agents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferably from about 30 to 150 liters perminute, and most preferably about 60 liters per minute. Aerosolscontaining greater amounts of medicament may be administered morerapidly.

The present invention also encompasses a method of treating infection byM. tuberculosis (e.g., slowing or inhibiting the growth thereof orkilling the bacteria), particularly drug resistant strains of M.tuberculosis (e.g., strains resistant to treatment with streptomycin,isoniazid, rifampin, ethambutol, or pyrazinamide) (see generally W.Jacobs et al., Science 260, 819 (7 May 1993); M. Baringa, New TestCatches Drug-Resistant TB in the Spotlight, Science 260, 750 (7 May1993); T. Frieden, New Engl. J. Med. 328, 521 (25 Feb. 1993)) andmultiple drug resistant strains of M. tuberculosis (e.g., strainsresistant to treatment with two or more of streptomycin, isoniazid,rifampin, ethambutol, or pyrazinamide), using lantibiotics. Lantibioticssuitable for use in the present invention include, but are not limitedto, duramycin, nisin, subtilin, epidermin, Pep 5, gallidermin,mersacidin, actagardine, cinnamycin, and ancovenin, or thepharmaceutically acceptable salt thereof, as discussed above. Duramycinand the pharmaceutically acceptable salts thereof are particularlypreferred. The tuberculosis infection may be in the lung of the subjectbeing treated, or may be an extrapulmonary infection such as of thepleura, lymphatic system, bone, genito-urinary tract, cerebral meninges,central nervous system, peritoneum, or skin.

Pharmaceutical compositions for use in the present method of treatingtuberculosis include those suitable for inhalation, oral, rectal,topical, (including buccal, sublingual, dermal and intraocular)parenteral (including subcutaneous, intradermal, intramuscular,intravenous and intraarticular) and transdermal administration.Inhalation therapy is discussed above. The compositions may convenientlybe presented in unit dosage form and may be prepared by any of themethods well known in the art. The most suitable route of administrationin any given case may depend upon the anatomic location of the M.tuberculosis infection in the subject, the nature and severity of thecondition being treated, and the particular active compound which isbeing used. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart.

In the present method of treating M. tuberculosis, the lantibiotics areadministered in a dose of from 1 to 100 mg/kg per day. The dose ofactive agent will vary according to the condition being treated and thedose at which adverse pharmacological effects occur. One skilled in theart will take such factors into account when determining dosage.

The present method of treating tuberculosis is concerned primarily withthe treatment of human subjects but may also be employed for thetreatment of other mammalian subjects, such as dogs, cats and cows, forveterinary purposes.

In one embodiment of the present invention, aerosolized duramycin isadministered to the lungs of a subject with pulmonary tuberculosis, inan amount sufficient to achieve dissolved concentrations of duramycin onthe airway surfaces of the subject of from 10⁻⁷ to 10⁻³ Moles/liter. Inanother embodiment of the present invention, duramycin is administeredby an intravenous bolus or by infusion, most preferably by infusion, ina dose range of from 70 mg/day to 2.5 g/day, and more preferably in adose range of from 150 mg/day to 2 g/day.

Lantibiotics used in the present method of treating tuberculosis may beadministered in conjunction with another anti-tubercular agent, suchother anti-tubercular agents including, but not limited to, ethambutol,streptomycin, isoniazid, rifampin, and pyrazinamide. These additionalanti-tubercular agents may be administered in a manner and in an amountin accordance with conventional techniques. See generally Goodman andGilman's The Pharmacological Basis of Therapeutics, 1210-1212 (SeventhEd. 1985).

In a further aspect of the present invention, lantibiotics may be usedalone or in combination with one or more anti-tubercular agents for theprophylaxis or treatment of tuberculosis.

In the manufacture of a medicament according to the invention (a"formulation"), active agents or the physiologically acceptable saltsthereof (the "active compound") are typically admixed with, inter alia,an acceptable carrier. The carrier must, of course, be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the patient. The carrier may be a solidor a liquid, or both, and is preferably formulated with the compound asa unit-dose formulation, for example, a tablet, which may contain from0.5% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention(e.g., the formulation may contain one or more additionalanti-tubercular agents as noted above), which formulations may beprepared by any of the well known techniques of pharmacy consistingessentially of admixing the components, optionally including one or moreaccessory therapeutic ingredients.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-inoil emulsion. Such formulationsmay be prepared by any suitable method of pharmacy which includes thestep of bringing into association the active compound and a suitablecarrier (which may contain one or more accessory ingredients as notedabove). In general, the formulations of the invention are prepared byuniformly and intimately admixing the active compound with a liquid orfinely divided solid carrier, or both, and then, if necessary, shapingthe resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder. Formulations for oral administration may optionally includeenteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit/dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, e.g., Pharmaceutical Research 3, 318 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe active compound.

Formulations suitable for inhalation administration comprise alantibiotic and optionally one or more additional anti-tuberculosisagents prepared as discussed above. Means for inhalation administrationare as discussed above.

The following examples are provided to more fully illustrate the presentinvention and should not be construed as restrictive thereof. In thefollowing examples, CF means cystic fibrosis, cm means centimeter, ccmeans cubic centimeter, kg means kilogram, ml means milliliter, M meansmolar, μM means micromolar, μg means microgram, μA means microampere,ED₅₀ means the dose which produces the desired effect in 50% of testanimals, and temperatures are given in degrees centigrade unlessotherwise indicated.

EXAMPLE 1 Effect of Duramycin on Chloride Conductance and CalciumMetabolism In Vitro

In vitro investigations were conducted employing primary cultures ofnormal and Cystic Fibrosis (CF) airway epithelium grown on collagensupports in defined medium to show the effect of duramycin on chlorideconductance and calcium metabolism.

1. Ussing Chamber Studies: The Cl⁻ secretory response to stimulation ofnormal and CF airway epithelium was studied under short circuit (I_(sc))conditions employing tissues mounted in Ussing chambers. Ussing chambersare well known in the art and the manner of their use in the presentexample would be readily apparent to one skilled in the art.

Tissues were first treated with amiloride to remove the Na⁺ current.Normal tissues were defined by demonstrating an increase in currentfollowing pretreatment with amiloride and subsequent treatment withforskolin (10 μM). The resulting increase in I_(sc) under theseconditions has been established as a Cl⁻ secretory response. In normaltissues and employing this protocol, subsequent use of duramycin at adose of 10⁻⁶ M induced a dramatic increase in the Cl⁻ current, raisingthe I_(sc) from a baseline of 20 μA/cm² after amiloride treatment to apeak of 50 μA/cm². (Data not shown.)

Primary cultures of normal tissues were treated with 10⁻⁶ M duramycinemploying three different bathing solutions in the Ussing chambers:control solution containing both Na⁺ and Cl⁻, solution without Na⁺, andsolution without Cl⁻. The tissues in both the control and Na⁺ freeconditions responded by increasing the I_(sc) after stimulation withduramycin. (Data not shown.) The tissue in the Cl⁻ free condition,however, showed a markedly attenuated response to duramycin. Theseobservations further support the conclusion that duramycin induces a Cl⁻secretory response in primary cultures of human airway epithelia.

The Cl⁻ secretory response to duramycin is characterized by a relativelysustained increase in I_(sc) lasting in some instances longer than 1hour. This is in contrast to the response to other agonists such as ATP,which produce a rapid onset of action and a rapid return to the baselinecurrent.

Duramycin-treated normal tissues demonstrated an increase in I_(sc) whensubsequently stimulated with ATP and forskolin. (Data not shown.) Thetissues ability to respond to these agents after duramycin treatmentdemonstrates that the tissues were not significantly damaged byduramycin.

2. Dose Response Study: A dose response study established that themaximal response to duramycin was achieved at 10⁻⁶ M with an ED₅₀ of3×10⁻⁷ M. Resistance across the tissue began to fall at doses of about2×10⁻⁶ M. The fall in resistance was interpreted as an indication oftoxicity.

3. Cl⁻ Response in Normal and CF Tissue: Seven normal and five CFtissues were studied, as above, employing duramycin at a concentrationof 10⁻⁶ M. These studies confirmed that a significant Cl⁻ secretoryresponse secondary to stimulation with duramycin could be produced inboth normal and CF tissues. The Cl⁻ secretory response in CF tissue(i.e., the change in I_(sc)) was approximately twice that seen in normaltissue. (Data not shown).

4. Mechanisms of Action: Preliminary studies were also doneinvestigating possible mechanisms of action. Employing positivecontrols, these studies were performed in a CF human transformed cellline, CFT43, to evaluate whether the tissue generated cAMP in responseto duramycin stimulation. The studies demonstrated that duramycinstimulation did not cause intracellular cAMP levels to rise (data notshown). Employing the same cell line, further studies were performed todetermine if the duramycin effect was mediated by the stimulation ofphospholipase C (PLC) and the generation of inositol phosphates (IPs).There were no detectable amounts of IPs produced in response tostimulation with duramycin (data not shown).

The in vitro studies described above indicate that (1) increases inI_(sc) in response to duramycin were secondary to Cl⁻ ion movementacross the tissue; and (2) duramycin stimulates a Cl⁻ secretory currentin both normal and CF tissues in vitro. Preliminary studies of themechanism of action of duramycin suggest that neither cAMP nor PLC isinvolved.

EXAMPLE 2 In Vivo Efficacy Animal Model

1. The Animal Model: The model developed employed 30 kg male dogs thatwere anesthetized with intravenous pentobarbital and maintained on aintravenous infusion of normal saline at 75 cc/hour. The dogs wereintubated. Ventilatory support was provided by a Harvard™ piston pump.Adequate ventilation and oxygenation were documented by arterial bloodgas studies. Peak airway pressures (PAP) were measured and continuousmonitoring of hemoglobin saturation was accomplished by pulse oximetry.The model allowed the animal to be absolutely stationary during thestudy, allowed ventilatory status of the animal to be fixed and set at aphysiological state, provided the opportunity for prolonged study of theanimal should this prove to be necessary, and standardized the amount ofstress the animals experienced.

Drug and vehicle were delivered through the endotracheal tube. The drugwas nebulized employing a Pulmo-Aid™ Compressor and a DeVilbiss™nebulizer. The drug (50 μM; 3 ml) in a saline vehicle or the salinevehicle alone (control) was nebulized for 6 minutes during which 0.7 mlthereof was delivered to the animal. Based on previous work, it wasestablished that the concentration of duramycin achieved in the airwaywas about 3×10⁻⁷ M, the estimated ED₅₀. After 6 minutes, a clean and drynebulizer was exchanged for the test nebulizer containing the drug orvehicle. The investigators were blinded as to whether drug or vehiclewas given.

2. Measurements of airway fluid: Airway liquid measurements were made byapplying dry preweighed filter paper to the cartilaginous portions ofthe airway for 20 seconds. This was accomplished employingtransbronchial biopsy forceps to grasp the papers. The forceps andpapers were stored in plastic catheters. Sampling the airway liquid wasaccomplished by first inserting the bronchoscope through theendotracheal tube. Care was taken not to allow contact between mucosaand the bronchoscope. The catheter containing the biopsy forceps andfilter papers was introduced into the airway lumen through the channelin the bronchoscope. The biopsy forceps were then pushed out of thecatheter and the filter papers gently applied to the mucosa. After 20seconds the biopsy forceps and filter papers were pulled back into thecatheter and removed through the bronchoscope for weighing. The filterpapers were accessed for weighing by again pushing the forceps holdingthe filter paper out of the catheter. In order to account forevaporative losses from the filter papers when exposed to room air, thetime required to place the papers on the scales and perform the initialweighing was recorded. Three subsequent weighings over time were thenmade. A regression line was calculated. The weight of the filter papersat time zero, that is when they were maximally wet, was defined as thetime when the filter papers were pushed out of the catheter after havingbeen applied to the airway mucosa. The amount of fluid on the filterpaper was taken as the difference between the dry weight of the papersand their weight at time zero. Each set of samples to be compared werefurther corrected for differences in the initial weight of the dryfilter papers.

3. Protocols: Two protocols were ultimately employed to accomplish theevaluation of the duramycin's effects on fluid transport, The goal ofthe first protocol was to study the effects on airway surfaces liquid of(1) repeatedly sampling the airway, and (2) nebulizing the normal salinevehicle. These issues were of concern in studying the duration of actionof duramycin since previous studies employing the awake sheep modeldemonstrated a marked secretory response in the airway, after foursamples had been obtained, and demonstrated a 35% increase in surfaceliquid after delivery of vehicle.

Protocol 1 required, immediately prior to conducting the test studies,weighing and loading the filter papers into catheters. Ten separatecatheter-forceps-filter paper set-ups were available. Ten measurementsof airway liquid were then made in different areas of the trachea over athirty-minute period, with measurements made at three minute intervals.The catheters were then reloaded and the saline vehicle nebulized ontothe airway. Ten more measurements of airway surface liquid were thenmade over another thirty minute period.

Protocol 2 required sampling the airway every four minutes to obtain 30measurements of airway liquid. This required reloading the 10 catheterstwice during each experiment. Each reloading required 20 minutes. Thisprotocol allowed the airway to be sampled over 160 minutes. Drug andvehicle administration were separated by one week in any one animal.Protocol 2 was employed to compare the effects of duramycin versusvehicle.

EXAMPLE 3 Results of In Vivo Efficiency Studies

Sampling the canine trachea ten times every three minutes (Protocol 1,above) did not significantly alter the amount of fluid recovered (datanot shown). Further, there was no difference between the amount ofliquid recovered from the trachea in the canine model at baseline andthat recovered after nebulization of the normal saline vehicle. FIG. 1.

Initial studies employing Protocol 1 to study the effects of duramycinon the airway demonstrated that duramycin caused a marked and sustainedincrease in the amount of fluid recovered from the airway. Afterduramycin was administered the amount of airway fluid received did notreturn to baseline during the 30 minute measurement period. Protocol 1thus did not allow a control comparison if the duramycin was givenfirst, since the airway surface liquid did not return to baseline eventwo hours following its administration.

In an attempt to deal with this unexpectedly long duration of drugaction, the 160 minute Protocol 2 was developed, which includesseparating the administration of drug and vehicle by one week. Animalstreated first with vehicle and then duramycin one week laterdemonstrated a stable baseline over 160 minutes. FIG. 2. Further, aseveral-fold increase in the amount of fluid recovered over baselineafter duramycin administration was observed. Sustained plateaus withouta significant slope were demonstrated for fluid measurements aftervehicle and after duramycin. Because there was no trend in the datasuggesting a flooding or drying of the airway, the values for the amountof surface liquid were averaged to produce a single value for duramycinand vehicle in each animal. This protocol too, however, was limited dueto the fact that one week following administration of duramycin, surfaceairway liquid measurements after vehicle administration remainedelevated as shown in FIG. 3. Therefore, only studies where vehicle wasadministered first were employed to compare the effects of duramycin tovehicle. Compared to the vehicle controls, these studies demonstrated asignificant (p=0.0179 (paired); p=0.0335 (unpaired)) increase in theamount of airway surface liquid recovered after duramycin treatment ofthe airway (FIG. 4). On average, duramycin produced a doubling of theamount of fluid recovered. This compared to the approximate 50% increasenoted in the sheep model after amiloride treatment. W. Mentz et al., Am.Rev. Resp. Dis. 134, 938-943 (1986).

EXAMPLE 4 Effect of Duramycin on M. tuberculosis Strains In Vitro

The effects of duramycin on the growth of three recent clinical isolatesof multidrug resistant Mycobacterium tuberculosis and one laboratorystrain of M. tuberculosis was assayed using the BACTEC™ 460 TB (BectonDickinson, Towson, Md.) system. The BACTEC™ 460 TB system is anautomated apparatus that assays the growth of microorganisms; the systemis known in the art and its use has been established in numerousclinical trials and cooperative studies. In the automated assay, agrowth medium (7H12 broth obtained from Becton Dickinson, Towson, Md.)containing ¹⁴ C-labeled palmitic acid as a single source of carbon isplaced in multiple 4 ml vials. Each vial is injected with dilutions ofthe drug or drugs being studied, and inoculated with 0.1 ml of bacterialsuspension from an actively growing BACTEC™ vial. Subsequent growth ofthe organisms leads to the metabolism of the substrate, with release of¹⁴ CO₂ into the sealed vial which is then measured by the BACTEC™ 460instrument. The data is expressed as a "growth index" (GI) on a scalefrom 0 to 1000. Test vials are read every day for 12 days and the growthcurve plotted (GI vs. day).

In the present experiment, each of three drug resistant strains of M.tuberculosis, and a drug-sensitive laboratory strain of M. tuberculosiswas tested against duramycin (100 μM, 10 μM, 1 μM, and 0.1 μM) andagainst four traditional anti-tuberculosis drugs: streptomycin (6μg/ml), isoniazid (0.2 μg/ml), rifampin (2 μg/ml), and ethambutol (7.5μg/ml).

M. tuberculosis 57778: M. tuberculosis 57778 is a recent clinicalisolate obtained from a New York City Hospital; this strain is resistantto streptomycin, isoniazid, and rifampin, and sensitive to ethambutol.FIG. 5 plots the growth curves for M. tuberculosis 57778, as describedabove, and indicates that 100 μM duramycin is equally as effective asethambutol against this ethambutol-sensitive strain of M. tuberculosis.

M. tuberculosis 63169: M. tuberculosis 63169 is another recent clinicalisolate obtained from a New York City Hospital; this strain is resistantto isoniazid and rifampin, and sensitive to streptomycin and ethambutol.FIG. 6 plots the growth curve for M. tuberculosis 63169 as describedabove, and indicates that 100 μM duramycin has activity against thisstrain approximately equal to that of the two drugs now known to beeffective against this strain (streptomycin and ethambutol).

M. tuberculosis 65021: M. tuberculosis 65021 is another recent clinicalisolate obtained from a New York City Hospital; this strain is resistantto streptomycin, isoniazid and rifampin, and partially resistant toethambutol. FIG. 7 plots the growth curve for M. tuberculosis 65021 asdescribed above, and indicates that 100 μM duramycin shows significantlymore activity against this strain than any of the four traditionalanti-tuberculosis drugs tested.

M. tuberculosis drug sensitive laboratory strain: M. tuberculosis(drug-sensitive laboratory strain) is sensitive to each of streptomycin,isoniazid rifampin, and ethambutol. FIG. 8 plots the growth curve for M.tuberculosis (drug-sensitive laboratory strain) as described above, andindicates that 100 μM duramycin is less active in vitro thanstreptomycin, isoniazid, rifampin, or ethambutol against this sensitivestrain.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

That which is claimed is:
 1. An aerosol pharmaceutical compositionconsisting essentially of, in a pharmaceutically acceptable carrier, alantibiotic which is a lanthionine-containing polypeptide or apharmaceutically acceptable salt thereof in an amount effective tohydrate lung mucous secretions,wherein said lantibiotic is selected fromthe group consisting of duramycin, nisin, subtilin, epidermin, Pep 5,gallidermin, mersacidin, actagardine, cinnamycin, and ancovenin; whereinsaid aerosol pharmaceutical composition comprises particles having aparticle size within the range of from about 0.5 to 10 microns; andwherein said pharmaceutical composition, when administered to an airwayfur face, comprises said lantibiotic in an amount sufficient to achieveconcentrations of said lantibiotic on said airway surface of from about10⁻⁹ moles/liter to about 10⁻³ moles/liter.
 2. An aerosol pharmaceuticalcomposition according to claim 1, further comprising amiloride in anamount effective to inhibit the reabsorption of water from lung mucoussecretions.